The CIpXP ATP-dependent protease from Escherichia coli provides an opportunity to explore the biophysical mechanisms of using chemical energy to facilitate unfolding and degradation. The CIpX component, which contains the substrate-recognition and ATP hydrolysis activities, acts as a chaperone in the absence of CIpP, the proteolytic component. The "molecular motor" model, suggested by previous work, proposes that CIpX unfolds substrates and presents them to the CIpP component. The primary goals of this work are to dissect the functional domains of CIpX through a combination of in vivo and in vitro studies and to test the following predictions of this model: subtrates that are energetically unstable should be easier to unfold and degrade rapidly, and that the protease complex binds the substrate at its C-terminus, then moves along the chain to the N-terminus. Point mutations in a CIpXP substrate will be made to test the effect of thermodynamic stability on proteolysis by the full-length protein. The "molecular motor" model will be directly visualized by attaching fluorescent labels to the substrate and the protease and observing active complexes with confocal microscopy. To understand the role of the N-terminal domain of CIpX in these activities, a series of N-terminal deletions will be tested for unfolding, ATP hydrolysis, substrate binding and proteolysis activity to ascertain the role this domain.